Production of membrane proteins in the milk of transgenic mammals

ABSTRACT

Production of proteins not normally secreted through conventional pathways such as membrane proteins including, for example, CFTR associated with cystic fibrosis, is now made possible by collection of such protein from the milk of lactating transgenic animals.

FIELD OF INVENTION

[0001] The present inventions involves the large-scale production ofmembrane specific proteins and more particularly involves the productionof such proteins using transgenic animals and most specifically involvesthe production of CFTR, the protein involved with Cystic Fibrosis.

BACKGROUND OF THE INVENTION

[0002] Striking progress has been made in our understanding of cysticfibrosis (CF) during the period since the gene associated with thedisease was identified¹⁻³. Many mutations within the gene have beenidentified in DNA from patients with CF⁴⁻¹⁰. The protein product of thegene, named CFTR, has been identified¹¹ and functional studies haveshown that CFTR cDNA is able to complement the defect in ion transportcharacteristic of cells from CF patients^(12,13). The domain structureof CFTR has been probed by analysis of mutated versions of theprotein¹⁴⁻¹⁸. Such studies indicate that CFTR is a Cl⁻channel^(14,19,20) and that it is required by phosphorylation of theR-domain^(16,18,21) and by nucleotide binding²². to our knowledge noneof such proteins have involved exogenous or recombinant membraneproteins.

SUMMARY OF THE INVENTIONS

[0003] In accordance with the principles of the present invention it hasbeen surprisingly discovered that exogenous or recombinant membraneproteins can be produced in the milk of transgenic animals. In the mostpreferred embodiment of the present invention, recombinant CFTR has beenproduced in the milk of transgenic mice containing CFTR cDNA downstreamof a mammary specific promoter. Most preferred embodiments will employlarger transgenic animals including, for example, rabbits, goats, sheepand cows which produce large quantities of milk however, the developmentof such animals takes considerably longer than the time periods requiredfor developing transgenic mice. While the time periods varyconsiderably, the procedures and methods are substantially identical.

[0004] Other embodiments include the apocrine like secretion in tissueculture of membrane proteins by cells which have been maintained in adifferentiated state. Such proteins would be secreted as part of themembrane released during the “pinching off” proces. Membrane proteinssuitable for production according to the methods of the present includereceptors, channels, viral glycoproteins, transporters and otherproteins typically associated with cellular membranes. Such membraneproteins can be used for therapeutics such as by administration of theabnormally deficient or missing protein, or by use as vaccines in thecase of viral glycoproteins including for example utilization of theenvelope of HIV, Herpes Virus or influenza in order to develop immunitywith respect to infection caused thereby.

[0005] Advantageously, methods of the present invention can also beemployed for the production of membrane proteins useful for diagnosticpurposes. For example, one could use the present methods to produce thereceptor for thyroid stimulating hormone (TSHR) which would be usefulfor the diagnosis of Graves Disease. Such proteins could also be used toscreen for therapeutically active compounds.

[0006] Preferred methods of the present invention will employ promoters,ideally coupled with suitable enhancers, that are mammary specific sothat production of the desired membrane protein is incorporated intomilk fat globules which will appear in the milk of a lactating femaletransgenic animal. Such an animal is, of course, by definition,reproductively competent. Greater appreciation of these and otheremodiments will be acquired by study of the following drawings, examplesand detailed procedures.

BRIEF DESCRIPTION OF THE FIGURES

[0007] Further understanding of the present invention may be had byreference to the figures wherein:

[0008]FIG. 1(a) shows the structure of the mammary specific expressionvector for producing CFTR using abbreviations: S, Sal I; R, EcoR I; N,Not I;

[0009]FIG. 1(b) shows identification of positive transgenic mice bySouthern blot analysis;

[0010]FIG. 2(a) shows the detection of CFTR in milk of transgenic mice;

[0011]FIG. 2(b) shows one-dimensional peptide analysis of CFTRsynthesized in recombinant C127 cells and in transgenic mice milk;

[0012]FIG. 3 shows the digestion of milk-derived CFTR with glycosidases;and

[0013]FIG. 4 shows fractionation of milk from transgenic mice; and FIGS.5A, B and C graphically show relevant vector constructions.

DETAILED DESCRIPTION AND BEST MODE

[0014] The 17.8 Kb DNA consruct used to produce transgenic mice capableof secreting CFTR in their milk is shown in FIG. 1A. It consists of afull length copy of CFTR cDNA (containing a point mutation at residue936 to inactivate an internal cryptic bacterial promoter that otherwiserenders the cDNA unstable¹⁵ ) inserted between exons 2 and 7 of the goatβ-casein gene. The β-casein gene contains a mammary gland specificpromoter which results in expression of the desired gene, e.g. CFTR orother membrane protein, within the mammary gland. Other milk specificpromoters which may be used in substitution include well known promoterssuch as ∝-casein, whey acid protein and β-lactoglobulin promoters. Mostpreferred constructs will include one or more enhancer elementstypically associated with such genes as has been described previously inthe art. Such promoters/enhancers are then associated with the codingsequence of the membrane protein of interest using conventionalrecombinant techniques. Such coding sequence may be either cDNA, partialor fully genomic DNA but more preferably either of the latter twocategories since such have been shown in transgenic systems to causegreater levels of expression. More particularly, the 4.5 kb Sal I-Sal Ifragment from pMT-CFTR¹⁵ was cloned into the Xho I site of CAS 1441 (seealso FIG. 5C). The cosmid vector CAS 1441 contains an altered goatβ-casein gene with an Xho I site in place of the coding portion of thegene. Earlier studies have characterized the goat casein gene and shownthat it directs the synthesis of a number of gene products in the milkof both transgenic mice and goats in a tissue-specific manner^(26,27).The portion deleted extends from the Taq I site in exon 2 to the PpuM Isite in exon 7.

[0015] With further reference to Figure series 5, a goat genomic DNAlibrary was constructed by cloning DNA fragments generated by partialMboI digestion of Saanen goat DNA into EMBL3 phage as described byManiatis et al. 31. The library, consisting of 1.2×106 recombinantphage, was screened with a 1.5 Kb HindIII/TthIII1 DNA fragment encodingthe entire mouse beta casein cDNA as described by Maniatis et al. 31.Three plaques designated 3-7, 3-8, and 3-11 exhibited the strongesthybridization signals under conditions of high stringency and each weresubjected to three rounds of plaque purification. Restriction enzyme andSouthern blotting analysis revealed that the inserts within recombinantphage 3-7 and 3-8 were subfragments of the 18.5 Kb insert contained inphage 3-11. Therefore, all DNA fragments used for construction of amammary gland specific expression vector were derived from clone 3-11which is shown in FIG. 5A. All subclones were constructed in pUC or pUCderivatives with modified polylinker.

[0016] To construct a mammary gland specific expression vector, theentire upstream (5′ portion) region from the SalI site to exon 2 and theentire downstream (3′ portion) region from exon 7 to the SalI site ofthe goat beta casein gene was used to direct expression to the mammarygland. To engineer the 5′ end of the beta casein gene, the TaqI site inexon 2 of clone Bc106 was replaced with a BamHI restriction site toproduce the plasmid Bc150 (FIG. 5A). The entire available 5′ region ofthe goat beta casein gene was constructed by the sequential addition ofthe subclones Bc104, Bc147, and Bc103 (FIG. 5A). The orientation ofsubclone Bc147 was verified by restriction analysis. The final vectordesignated Bc113 contains a SalI site at the 5′ end and a BamHI site atthe 3′ end. The downstream BamHI was subsequently changed to an XhoIsite to form Bc114.

[0017] The 3′ end of the goat beta-casein gene was constructed in asimilar fashion to the 5′ end. The 1.8 Kb Bc108 clone (FIG. 5A) wasdigested with PpuMI to allow for the addition of a BamHI linker. TheBamHI/HindIII fragment from the engineered clone Bc108 was ligated intothe vector Bc109 which contained the extreme 3′ flanking region of thegoat beta casein gene. The entire 3′ end was completed by the additionof the 4.4 Kb HindIII fragment from Bc108 was cloned into the modifiedBc109 vector and screened for orientation. The new vector was designatedBc118 and contained the entire 3′ region of beta casein from exon 7through the poly A signal and 5 Kb downstream to the SalI site of EMBLclone 3-11. Subsequently, an XhoI site was introduced at the 5′BamHIsite and a NotI site at the 3′ SalI site of Bc118 to produce the vectorBc122.

[0018] The overall cloning strategy followed to construct the betacasein CFTR vector (Bc8) is shown in FIG. 5B and 5C. To construct a goatbeta casein vector containing all the available upstream and downstreamsequences, a 1.7 Kb XhoI to SalI stuffer fragment containing a uniqueSacII site cloned into the XhoI site of Bc114 and Bc122 to form vectorsBc143 and Bc144, respectively. The complete vector was made by cloningthe SalI to SacII fragment from Bc143 into a SalI to SacII cut Bc144 toform the plasmid Bc6. The stuffer fragment was removed from the plasmidby digestion with BamHI and a unique XhoI site was inserted to form thevector Bc145. The SalI to NotI fragment from Bc145 which contains the 5′and 3′ beta casein sequences was subcloned into the cosmid clone CAS1438to form the cosmid CAS1441. CAS1438 is a cosmid vector constructed inpHC79 containing an engineered SalI and NotI site and 21 Kb of 5′flanking sequence from the Bovine alpha-casein gene³² (Meade et al.Biotechnology 8, 443-45 1990). The expression vector Bc8 was constructedby digesting the vector CAS1441 with XhoI and ligating in the 4.5 KbSalI fragment from pMT-CFTR^(11,17). Orientation of the CFTR cDNA wasconfirmed by restriction analysis. To obtain a fragment formicroinjection, the vector Bc8 was digested with SalI and NotI torelease the beta casein CFTR portion of the vector for purification. Thefinal microinjection fragment contained 4.2 Kb of 5′ flanking sequence,exon 1, intron 1, a portion of exon 2, 4.5 Kb CFTR cDNA, a portion ofexon 7, intron 7, exon 8, intron 8, exon 9, and 5.3 Kb of 3′ flankingsequence. This fragment from Bc8 was injected into mouse embryos andreimplanted using standard methods. Transgenic mice were identified bySouthern blot analysis of DNA from the tails of resulting offspring.

[0019] To screen for positive transgenic mice, approximately 15 μg ofgenomic DNA from each founder mouse was digested with EcoR I, separatedon a 1% agarose gel, transferred to magnagraph nylon (Fisher Scientificand hybridized with the 384 bp Pvu II-Pvu II fragment from exon 7 of themouse β-casein cDNA³⁰ and the 4.5 kB SaI I-SAI fragment of pMT-CFTR.Copy number was quantitated using a Betascope 603 Analyzer.

[0020]FIG. 1B shows DNA from the positive founder animals. Band I ismouse β-casein DNA and bands II, III and IV are CFTR DNA. Lane 1 is acontrol mouse and lanes 2 to 5 show results of four founder transgenicmice. Such analysis indicates the copy number of the transgene in thelines varied between 1 and 10.

[0021] Founder mice were bred to produce lactating transgenic females.Milk from such animals was collected, diluted with PBS (phosphatebuffered saline) and treated with RIPA (50 mM Tris-HCI [pH 7.5], 150 mMNaCI, 1% Triton X-100, 1% sodium deoxycholate and 0.1% sodium dodecylsuplhate) buffer to solubilize membrane proteins. Briefly, samples wereexamined for the presence of CFTR by immunoprecipitation with pAbEx13, ahighly purified polyclonal antibody raised against an R-domain fusionprotein (see Canadian Patent Application 2,037,478-1, fully incorporatedherein by reference), followed by in vitro phosporylation treatment ofthe washed immunoprecipitate with protein kinase A(PKA) in the presenceof [_(y) ³²P]ATP^(11,15). More specifically, procedures for preparingcell lysates immunoprecipitation of proteins using pAbEx13,one-dimensional peptide analysis and SDS-polyacrylamide gelelectrophoresis were as described by Cheng et al. ^(15,16). In vitrophosphorylation of the CFTR-containing immunoprecipitates was achievedby incubating with protein kinase A and [_(y) ³²P]ATP (10 μCi) in afinal volume of 50 μl in kinase buffer (50 mM Tris-HCI, pH 7.5, 10 mMMgCl₂ and 100 μg ml⁻¹ bovine serum albumin) at 30° C. for 60 min. Milkwas collected from the transgenic mice at days 3, 5, 7 and 9 oflactation. Mice were injected with oxytocin (2.5 IU) and milked 5 minlater with a suction device. The milk was diluted (10 fold) andsolubilized in RIPA buffer prior to immunoprecipitation.

[0022]FIG. 2A shows a polyacrylamide gel of samples from milk of twofemales of line #12 and a control mouse. Lane 1 contains animinunoprecipitate from approximately 3×10⁶ recombinant C127 cellsstably transfected with a wild type CFTR cDNA. Immunoprecipitates of 50μl of control milk (lane 2), milk from transgenic mouse #12-6 (lane 3)and mouse #12-9 (lane 4) were subjected to the same treatment. Thepositions of the fully processed CFTR (band C) and the partiallyglycosylated form (band B) are indicated on the right margin. Exposuretime was 30 min at −70° C. As can be seen from the results, a diffuseband of labeled protein is detected in the milk from both of thetransgenic mice but not the control. The protein (lanes 3 and 4)migrates slightly more rapidly than the fully processed version of CFTR(lane 1) from stably transfected mouse C127 cells containing a BPV-CFTRvector. Such differences in migration of CFTR have been detectedpreviously in samples of the protein from various recombinant and highproducer cells. They have been shown to be caused by differences inglycosylation^(15,20).

[0023] To confirm that the protein detected in FIG. 2(a) is indeed CFTR,the labeled protein was eluted and subjected to partial proteolyticcleavage using Staphylococcus aureus V8 protease followed bypolyacrylamide gel analysis. Specifically, the vitro labeled bands Cfrom recombinant C127 cells (lanes 1-3) and from mouse #12-6 (lanes 4-6)were excised from the gel and digested with different amounts ofStaphylococcus aureus V8 protease^(11,15). Proteins in lanes 2 and 5were digested with 0.17 μg μg⁻¹ S. aureus V8 protease and those in lanes3 and 4 with 0.017 μg μl⁻¹ enzyme. Lanes 1 and 6 were untreated samples.Exposure time was 24 h at −70° C. The results depicted in FIG. 2(b) showthat the milk derived protein yields a fingerprint indistinguishablefrom that of CFTR derived from C127 cells. Thus, the milk from line #12mice contained authentic CFTR. Similar analysis of milk from the othertransgenic mouse lines for the presence of CFTR showed that line #38contained CFTR but in lesser amounts than in line #12 whereas lines #18and #13 contained no detectable protein.

[0024] To confirm that the difference in mobility between milk-derivedand C127 cell-derived CFTR results from different patterns ofglycosylation, the ³²P-labeled proteins were subjected to digestion withN-Glycanase® enzyme, endoglycosidase H and endoglycosidase F. Inparticular, the CFTR immunoprecipitated from the milk of transgenicmouse #12-9 and from recombinant C127 cells were phosphorylated in vitrousing protein kinase A and [_(y) ³²P]ATP. Following elution frompolyacrylamide gels, the CFTR proteins were either subjected to notreatment (lanes 1,3, 5 and 7) or were incubated with N-Glycanase (lanes2 and 4), endoglycosidase H (lane 6) or endoglycosidase F (lane 8).Samples were separated by electrophoresis and analyzed byautoradiography. The positions of the fully processed form of CFTR (bandC) and the non-glycosylated version (band A) are indicated on the rightmargin. Exposure was for 24 h at −70° C. The enzymes N-Glycanase,endoglycosidase H and endoglycosidase F were from Genzyme Corp.Conditions for digestion with the respective glycosidases were asspecified by the manufacturer, except that incubations were performed at37° C. for 4 h only. CFTR was immunopurified using the polyclonalantibody pAb Ex13, phosphorylated in vitro with protein kinase A and[_(y) ³²P]ATP, separated on SDS-polyacrylamide gels, and then extractedfrom the gels by macerating the gel pieces in protein extraction buffer(50 mm ammonium bicarbonate, 0.1% sds and 0.2% β-mercaptoethanol).Eluted proteins were recovered by trichloroacetic acid precipitation.

[0025] The results depicted in FIG. 3 show that milk CFTR is digested byN-glycanase to yield a band of greater mobility (lane 2) thatco-electrophoreses with the corresponding band from C127 cell-derived,N-glycanase-treated CFTR (lanes 4). This band consisted ofnon-glycosylated CFTR^(11,15). As shown previously for COS-7 derivedCFTR¹⁵, milk derived protein is resistant to cleavage by endoglycosidaseH or F (lanes 6 and 8). It is therefore clear that the protein detectedin the milk of transgenic mice is a fully glycosylated form of CFTR thatcontains carbohydrate side chains of somewhat less complexity than CFTRfrom recombinant C127 cells. In separate studies tissue plasminogenactivator (tPA) produced in recombinant cells has been compared withthat produced in milk of transgenic goats and likewise shown to havedifferences in the pattern of glycosylation²⁶.

[0026] We surprisingly discovered that membrane proteins such as CFTRcan be expressed in milk. We discovered that CFTR is present in themembrane of milk fat globules and thus is found in the lipid-richfraction of the milk, that is, in the cream. To isolate the CFTR, milkfrom transgenic animals was fractionated as described by Imam et al. andPatton & Huston²⁹. In summary, milk was first centrifuged at 3000 g toseparate the cream which floats from the skim milk. The bulk of thecaseins were separated from the skim milk by further low speedcentrifugation at 12,000 g. Since milk fat globules are fragile, somemembrane fragments remain in the supernatant of centrifuged skimmilk^(23,29). Such residual membrane vesicles and fragments werecollected by further high speed centrifugation at 100,000 g. In detail,0.4 volume of wash buffer (10 mM Tris-HCI, pH 7.5, 0.25 M sucrose and 1MM MgCI₂) was added to whole milk (approximately 300 μl) and centrifugedat 3000 g for 15 min at room temperature to separate the cream. Theresulting skim milk was siphoned off by inserting a needle through thelayer of cream. The cream was washed twice with approximately 5 volumesof wash buffer and dispersed in RIPA buffer. The siphoned skim milk wascentrifuged at 12,000 g for 10 min to pellet the caseins. The caseinpellet was washed once with 5 volumes of wash buffer and resuspended inRIPA buffer. Membranes and vesicles contained in the resultant skimsupernatant were collected by further centrifugation at 100,000 g for 2h.

[0027]FIG. 4 shows PKA immunoprecipitation assays of the variousfractions obtained. Whole milk (lane 1), the cream fraction (lane 2),the casein-containing pellet (lane 3), the 100,000 g skim pellet (lane4) and the 100,000 g skim supernatant (lane 5) were solubilized withRIPA buffer, immunoprecipitated with pAb EX13 and phosphorylated invitro using protein kinase A and (_(y) ³²P)ATP. Exposure time was 1 h at−70° C. About 65% of the CFTR was present in the cream (lane 2) and theremaining CFTR was in the high speed pellet from the skim milk fraction(lane 4). Virtually no CFTR was present in the casein fraction nor inthe high speed supernatant.

[0028] The data shown hear clearly indicates that authentic CFTR can beproduced in the milk of transgenic mice. The form of protein producedrepresents mature fully glycosylated CFTR rather than a partiallyglycosylated intracellular intermediate as indicated by itsinsensitivity to endoglycosidase H but sensitivity to N-glycanase. Thispattern of sensitivity implies CFTR has a carbohydrate structureconsistent with it having traversed both the endoplasmic reticulum andthe Golgi, but why is it present in the milk ?

[0029] This surprising and unexpected result must occur because some,perhaps all, mature CFTR must reach the apical plasma membrane ofmammary epithelia in lactating animals. This epithelia is activelyinvolved in high level secretion, not only of soluble milk proteins suchas the caseins through the normal protein secretion pathway, but also oflipids in the form of fat globules^(23,24). It has been discoveredtherefore that CFTR is secreted by apocrine like secretion into the milkrather than remaining in the cellular membrane. If the CFTR is notsecreted as a soluble protein, but as part of the milk fat globule. Suchglobules are composed of an outer covering of pinched-off plasmamembrane, a small amount of underlying cytoplasm comprising theso-called cytoplasmic crescent and a globule of fat^(23,24). WhetherCFTR is incorporated into the fat globule membrane by a random processof plasma membrane blebbing or whether it is actively recruited to theexiting vesicle as it pinches off remains unknown. The demonstrationthat most CFTR in milk floats away from the bulk of milk proteins uponcentrifugation, and the remainder separates from casein and can besedimented at high speed strongly suggests that CFTR is present inmembrane structures some of which include fat globules. The mostplausible explanation of the results is that the bulk of CFTR exits thecell in the membrane of milk fat globules and that some of theseglobules break off membrane vesicle fragments upon handling.Alternatively, perhaps some empty membrane vesicles containing CFTR butno fat globules are directly shed from the mammary epithelia.

[0030] As a result of the present discovery that CFTR can be sequesteredinto milk, it will be readily apparent to those skilled that other humanmembrane-associated proteins such as receptors (e.g. cytokine receptors,hormone receptors), viral glycoproteins, transporters and channels thatsort at least in part to the apical surface may be produced by thetransgenic methods of the present invention. Alternatively, suchmembrane related proteins can be produced in vitro such as by tissueculture of appropriate cells capable of apocrine secretion. Mammarycells maintained in a differentiated state would be the preferredembodiment of such an approach. Although any cell capable of blisteringor “blebbing-off” membrane associated proteins such as those associatedwith apocrine secretion will suffice. Previous demonstration that thesynthesis of soluble human proteins in transgenic milk can be scaled upto production in transgenic goats^(26,27) confirms that virtuallyunlimited amounts of membrane proteins could be produced by this means.

[0031] The application of the present invention to CFTR production willenable the development of new therapies for CF. Of direct implication isthe capability to now produce sufficient CFTR protein to allowtherapeutic treatment of CF patients by protein replacement. In suchtherapy, CFTR protein is administered to the airway cells, preferably byaerosolization and inhalation, typically in conjunction with helperagents to assist in delivery the CFTR to the airway cell surfacesthrough the mucous layer for incorporation into the airway cellmembrane. Because there is turnover of such cells, treatment by thismodality is a continuous prospect.

[0032] Other exciting opportunities now enabled by the present inventioninclude protein replacement therapies for other defective or deficientmembrane protein mediated diseases as well as diagnostic and screeningassays previously made impossible due to the absence of commerciallyviable membrane protein production systems since membrane proteins arenot secreted into the surrounding medium. For example, the ability tonow produce identified and sequenced cytokine receptors allows one tocreate not only an assay with a substrate (using either purifiedreceptor or the milk fat globule itself or membranes therefrom havingthe receptor exposed) specific for the cytokine (whose binding to thesubstrate could be readily detected by a labeled, cytokine specificantibody) but can also be used as a screening device to detect otherproteins or peptides capable of binding to the same receptors. Suchpeptides might serve to activate the receptor or alternately asantagonists to prevent cytokine function. Selection of new effectivedrugs would be facilitated using such a screening approach. Similarly,these techniques would be equally useful for hormone receptors such asthose specific for thyroid stimulating hormone as earlier mentioned.

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What is claimed is:
 1. A method for producing a membrane proteincomprising the steps of: (a) inserting into a mammalian embryo a DNAconstruct comprising a mammary gland specific promoter and a DNAsequence encoding said membrane protein, (b) allowing said embryo tomature into a reproductively competent mammal, (c) inducing lactation infemales of said reproductively competent mammal or in female progeny ofsaid reproductively competent mammal, and (d) collecting the milk ofsaid lactating female which contains said membrane protein.
 2. Themethod of claim 1 wherein said mammary gland specific promoter isselected from the group of genes consisting of betalactoglobulin, alphacasein, beta casein and whey acid protein encoding genes.
 3. The methodof claim 2 wherein said membrane protein is CFTR.
 4. The method of claim3 further comprising the step of isolating the CFTR from the collectedmilk.
 5. The method of claim 2 wherein said membrane protein is selectedfrom the group consisting of hormone receptor proteins, cytokinereceptor proteins, viral glycoproteins and transmembrane channels. 6.The mammalian embryo produced by the method of claim
 1. 7. The mammalproduced by the method of claim
 1. 8. The membrane protein produced bythe method of claim
 1. 9. A method for producing a membrane proteincomprising collecting milk comprising said membrane protein from atransgenic mammal having in its genome a gene encoding said membraneprotein and under the control of a mammary gland specific proteinpromoter.
 10. The method of claim 9 wherein said mammary gland specificpromoter is selected from the group of genes consisting ofbetalactoglobulin, alpha casein, beta casein and whey acid proteinencoding genes.
 11. The method of claim 10 wherein said membrane proteinis selected from the group consisting of hormone receptor proteins,cytokine receptor proteins, viral glycoproteins and transmembranechannels.
 12. The method of claim 10 wherein said membrane protein isCFTR.
 13. The method of claim 12 further comprising the step ofisolating the CFTR from the collected milk.
 14. A method for producing amembrane protein comprising culturing cells transformed with a geneencoding a membrane protein and capable of apocrine secretion andcollecting said membrane protein from the cell culture media.
 15. Themethod of claim 14 wherein said membrane protein is selected from thegroup consisting of hormone receptor proteins, cytokine receptorproteins and transmembrane channels.